JP6723541B2 - Imaging microfluidic flow cell assembly and method of use thereof - Google Patents

Description

The present invention relates to imaging microfluidic flow cell assemblies and methods of use thereof.

The present invention is generally a microfluidic that encapsulates a sample and allows for subsequent controlled delivery of reagents to the sample, eg, analysis of a mounted biological sample using multiple in situ biomarker staining and dye cycling. Related to the making and use of flow cell subassemblies.

For multiplex in situ biomarker analysis, tissue samples or tissue microarrays (TMA) mounted on glass slides are stained with multiple molecular probes to quantitatively or qualitatively investigate biomarker expression or spatial distribution There is a need. The staining and data collection steps are usually performed using error-prone, time-consuming manual techniques. After staining, a coverslip needs to be placed over the sample to keep it wet during subsequent imaging (data collection). The coverslips should then be removed before the next staining round. This process of placing and removing the coverslip can result in sample loss or sample migration on the glass slide, which confuses downstream analysis. Staining is generally performed by applying a staining reagent to the sample and allowing the staining reagent to stand for a predetermined incubation period. Therefore, the staining time is determined by the molecular diffusion of the staining component from the bulk solution into the sample. The method of actively mixing the reagents on top of the sample during the incubation is aimed at ensuring uniform staining throughout the sample and facilitating the interaction between the staining components and the sample. Such methods rely on the induction of bulk fluid movement, which does not have fluid separation regions that affect staining uniformity, and thus has a lower reagent volume limit.

Therefore, there is a need for a system that can automate the workflow of in situ multiple biomarker analysis while providing optimal conditions for reagent delivery and data collection. One way to control the reagent delivery of small volumes of reagents is to limit the reagents to the area close to the sample by using fluid channels. The diffusion length is determined by the height of the channel, allowing fresh (well mixed) reagent to flow through the channel to maintain optimal reagent concentration near the sample.

Generally, microfluidic flow cells comprise one or more sealing layers sandwiched between two substantially flat base layers. This sealing layer creates the shape of the fluid channel, forms part of the channel wall, and usually defines the thickness of the channel. This sealing layer can be formed by cutting or molding the outside of a solid material into a defined shape, or by printing a solidifying liquid material on one substrate. The two substrates enclose the fluid channels and act as upper and lower channel walls. The leak tight seal is made by clamping the sealing layer between the substrates and/or by adhering the sealing layer to one or both of the substrates.

Furthermore, the construction of the flow cell indicates that there are limitations to the way in which the encapsulated sample can be detected and analyzed through traditional imaging equipment. In particular, there is a need for a method of imaging a sample through a flow cell that provides a means for focusing, quality adjusting, and aligning the flow cell with the objective lens of the imaging device of the captured image.

Accordingly, there is a need for a microfluidic flow cell that allows a wide range of substrate materials and facilitates access to imagers and methodologies while not needing to be fluidly connected by any substrate.

US Patent Application No. 2013/0287645

The present invention overcomes the aforementioned shortcomings by providing a microfluidic subassembly that can be assembled in a flow cell with fluidic connections outside the primary substrate.

In accordance with one aspect of the present invention, a microfluidic subassembly having a laminated planar assembly is disclosed. The laminated planar assembly comprises an adhesive layer, a transparent substrate layer and a gasket layer where each layer is adhered to each other, the adhesive layer and the gasket layer extending beyond the extent of the substrate layer. In addition, there are one or more fluid ports located outside the boundaries of the substrate layer. By construction, the substrate layer bends in either direction to form a flexible transparent lid that can change the internal dimensions of the subassembly.

In accordance with yet another aspect of the invention, there is disclosed a microfluidic flow cell comprising the above subassembly, further comprising a solid support adhered to the microfluidic flow cell subassembly.

According to another aspect of the invention, a method of analyzing a biological sample bound to a solid support using the described microfluidic flow cell is disclosed. The method comprises adjusting the quality of the captured image of the sample carried on the solid support of the flow cell by aligning the flow cell with the objective lens of the imager, and adjusting the amount of fluid in the flow cell. Flexing the flexible lid within a range of about ±200 μm from its center point.

Various other features and advantages of the present invention will be made apparent from the following detailed description and the drawings.

These drawings illustrate embodiments presently contemplated for carrying out the invention.

Schematic view of a representative subassembly showing a laminated planar assembly (A), schematic view of a representative subassembly showing a laminated planar assembly (B), seen from the gasket side, adhesive layer side, before assembly FIG. 3C is a schematic view (C) of an exemplary subassembly showing a planar assembly. Schematic view (A) of an assembly flow cell (160) with a subassembly (100) adhered to a solid support (150), prior to assembly, as seen from the subassembly side, adhered to the solid support (150). Schematic (B) of assembled flow cell (160) with subassembly (100), assembled flowcell (160) with subassembly (100) adhered to solid support (150), viewed from the side of the solid support. () is a schematic view (C). FIG. 6 is a schematic view of a connector assembly showing a laminate layer (300) and assembly fixture (350) forming a thin film connector (360). Schematic (A) depicting a thin film connector first bonded to a flow cell gasket material (410) as part of a subassembly (420), a thin film first bonded to a flow cell gasket material (410) as part of a subassembly (420) FIG. 6B is a schematic diagram (B) in which a connector is adhered to a solid support (430) to form a microfluidic flow cell (440). FIG. FIG. 6 is a schematic diagram showing a thin film connector configured with an incorporated reagent well (510) and valve (520). Schematic diagram showing a fluid module arranged with bent, thin-film fluid connections (A), schematic diagram showing a fluid module arranged with twisted, thin-film fluid connections (B), arranged in series, multiple modules FIG. 6C is a schematic diagram (C) showing fluid modules arranged with thin film fluid connections stacked to enable. FIG. 3 is two schematic diagrams showing that the gasket layer can be a valve in the assembly flow. FIG. 6 is a schematic diagram showing how a fluid valve is closed by deforming a gasket layer. 1 is a schematic view of an embodiment in which a fluid connection fitting comprises a ring or sealing device with height. FIG. 6 is a schematic diagram showing how a microfluidic flow cell contacts a raised sealing surface (190) to form a seal against a gasket layer. FIG. 6 is a schematic view of an assembled flow cell using another design of gasket and adhesive layer. FIG. 9A is a view of one of the elevated sealing devices showing gasket (130) and ledge (195). FIG. 9B shows that the flow channel can be formed with the gasket layer (130) as the bottom surface. FIG. 4 is a diagram showing the results of imaging a human tissue section with an objective lens at a magnification of ×20, showing successive images at various time points from a convex overfilled flow cell of 50 μm to a concave underfilled flow cell of about −50 μm. Show. It is a process flow diagram showing one workflow candidate for DNA FISH automation in a flow cell. FIG. 12A shows an image of a magnetic auxiliary fluid connection consisting of a snug fit small inner diameter tubing and an inner bond of an annular magnet, FIG. 12A showing a cylindrical magnet placed on the opposite side of the thin film connector, and FIG. FIG. 6 illustrates how magnets are also used to block ports and seal chambers. FIG. 6 is a photomicrograph of a successful DNA FISH experiment in a microfluidic flow cell showing DAPI, CEP17 and Her2 staining.

In order to more clearly and concisely describe the claimed subject matter, the following definitions are provided for certain terms used in the following description and appended claims.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein throughout the specification and claims, the approximate term (Approximating language) modifies any quantitative expression that can be varied to the extent that it does not change the associated basic function. Can be applied to Thus, values modified by terms such as "about" are not limited to the exact values specified. Unless otherwise specified, all numbers used in the specification and claims to describe amounts such as raw materials, properties such as molecular weight, reaction conditions, etc. are all modified by the term "about". Should be understood as being done. Therefore, unless indicated to the contrary, the numerical parameters recited in the above description and the appended claims may vary depending on the desired properties sought to be obtained by the present invention. It is a value. At a minimum, each numerical parameter should be construed by the application of ordinary rounding techniques, taking into account at least the reported significant figures.

As used herein, the term "biological sample" refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid of in vivo or in vitro origin. Such samples may be whole cells, tissues, fractions and cells isolated from mammals, including, but not limited to, humans, whole or part blood samples and other biological fluids. The biological sample may be mounted or fixed on a solid support, for example, a tissue section mounted on a microscope slide, a tissue microarray or a blood smear, or a subassembly on the solid support. It may be introduced into the flow cell after being adhered.

As used herein, the term "consumables" refers to disposable parts designed for single use or limited use. In some situations, consumables may have a useful life that is less than the useful life of the system in which they are used; in other situations, consumables are parts that separate from the system in which they are intended to be used. May be stored and manufactured.

In certain embodiments, the microfluidic subassemblies are sequentially stained with dye and imaged with any high-resolution microscope to inactivate or inactivate the fluorescent reporter, after which the cycle is repeated. , Providing means for encapsulating a biological sample. As used herein, a microfluidic subassembly may also be referred to as a microfluidic chamber that creates a chamber at the center of the assembled flow cell. As used herein, microfluidic subassemblies may also be referred to as subassemblies. In certain embodiments, the subassembly is a consumable such that the subassembly is designed for single use or limited use.

The subassembly device provides a means of enclosing the sample within the chamber. In certain embodiments, the sample is a biological sample mounted on a solid support, such as a standard microscope slide, and the sample is maintained in a controlled environment during subsequent processing steps. The biological sample may be placed on a solid support and then encapsulated in a subassembly device that results in the formation of a flow cell. In certain embodiments, biological samples can include, but are not limited to, whole cells, tissue sections, tissue microarrays or blood samples. In certain embodiments, the tissue section may be a fixed tissue sample. In certain embodiments, the flow cell comprising the subassembly may be a consumable such that the subassembly is permanently bonded to a solid support for real-time or near real-time analysis. In certain embodiments, the assembled flow cell may serve as an archive of samples for future testing or analysis.

The biological sample is made by flowing the biological sample into the cell after the formation of the flow cell, and using chemical or biological means, electrostatic interaction, non-specific adsorption, dielectrophoretic force, magnetic force, optical tweezers, physical by microstructure. It can also be attached to a solid support by capturing a biological sample by dynamic capture or similar means. In certain embodiments, the flow cell containing the encapsulated sample may be stored as is for subsequent analysis.

In one embodiment, the microfluidic flow cell formed using the subassembly need not be physically clamped to maintain a seal to the solid support, or maintain the integrity of the seal.

In certain embodiments, the contents of the flow cell can be analyzed in situ using various optical, electrical, magnetic or electromechanical devices in communication with the flow cell. In another embodiment, the substance can be transported out of the flow cell for subsequent analysis. In one embodiment, the flow cell is used for multiple tissue staining and imaging as described in US Patent Application No. 20099253163 and US Patent No. 7629125. In yet other embodiments, the flow cell can be used in combination with techniques involving morphology with or without other cell analysis techniques or extraction methods. Analytical techniques include, but are not limited to, DNA analysis or amplification, RNA analysis or amplification, nucleic acid sequencing, protein analysis, antigen retrieval, hematoxylin and eosin staining (H&E), immunofluorescent staining (IF), Mention may be made of immunohistochemical staining (IHC), fluorescent in situ hybridization (FISH) or other histological and morphological staining techniques.

Representative embodiments of subassemblies are shown in FIGS. 1A and 1B and 1C. The subassembly device (100) comprises a laminated planar assembly comprising an adhesive layer (110), a substrate layer (120) and a gasket layer (130), the gasket layer and the adhesive layer extending beyond the substrate layer. .. These layers adhere together and have holes located along the outer boundaries of the adhesive layer and the gasket layer to form fluid inlet/outlet ports (140). Fluid inlet/outlet ports may also be referred to herein as ports. 1B shows the subassembly as seen from the gasket (130) side, and FIG. 1C shows the subassembly as seen from the adhesive layer (110) side.

The substrate layer (120), in certain embodiments, the substrate material may be composed of glass, but may also be composed of plastic, metal, silicon, ceramic, insulator, silicone or combinations thereof. In certain embodiments, the preferred material is glass or the substrate is a cover glass slip. In certain embodiments, for example, where the substrate is a coverslip, the coverslip is made of glass, such as silicate or borosilicate glass, or polyolefin, and is NUNC™ brand with precise light transmission. It may consist of specialty plastics such as Thermanox® coverslips. Quartz glass coverslips can also be used if UV transmission is required, for example for fluorescence microscopy.

The subassembly (100) can be adhered to a solid support (150) as shown in FIG. In the example of FIG. 2, a tissue microarray is mounted on a solid support. Efficiently adhering the subassemblies forms a sealed microfluidic flow cell (160) along the entire boundary. This is further illustrated in Figures 2B and 2C, which show the assembled flow cell from both sides, with Figure 2B being the flow cell from the gasket side and 2C from the solid support side. In some embodiments, the total thickness of the subassembly can be designed to be about 25 to about 1200 μm, the thickness being between about 20 and about 1000 μm gasket and about 5 to about 200 μm. The combined thickness of the adhesive layers in between.

In certain embodiments, one or both of the substrates (120) and the solid support (150) are transparent at a particular range of wavelengths. Therefore, optical analysis of materials/structures within the flow cell can be accomplished either by epi-illumination or by transillumination when both are transmissive. In embodiments where the assembled flow cell can be used for multiple tissue staining and analysis, the use of both a transparent substrate and a solid support allows both epifluorescence imaging and transmission brightfield imaging. This enables fluorescence-based molecular pathology and conventional brightfield imaging based on, for example, diaminobenzidine (DAB) staining or hematoxylin and eosin dye (H&E) chromogenic staining.

As used herein, the term "glued together" or "gluable" refers to the joining together of parts or materials to form a seal at the mating surfaces of the materials. Adhesion refers to the use of chemical adhesives to form bonds, including, but not limited to, silicones, epoxies, acrylics, room temperature curable materials (RTVs), thermoplastics or These combinations may be mentioned. Adhesion can also be achieved by overmolding one material over another to create a seal by mechanical or chemical interaction at the interface of the two materials. In certain embodiments, adhesion can be achieved through the application of pressure, temperature or external conditions such as exposure to light or radiation. Adhesion can provide a bond to the mating surface that is so strong that cohesive failure occurs upon separation. In other cases, adhesion may result in a bond that can be broken with minimal force such that the bond surface can be repositioned or the bond is considered a temporary bond.

In certain embodiments, the solid support carries an immobilized biological sample such that the sample is enclosed in the resulting flow cell. The fluid inlet/outlet port (140) is configured to extend beyond the substrate (120) such that no through holes are required in either the substrate (120) or the solid support (150).

In certain embodiments, the adhesive material is adhesive, including but not limited to chemical adhesives such as silicone, acrylics, epoxies, room temperature curable materials (RTVs), thermoplastics, or combinations thereof. Including. In certain other embodiments, the adhesive material may be adhesive tape, silicone, thermoplastic elastomer, paraffin wax, printed adhesive material or plastic film. In yet other embodiments, the adhesive material may be a light, heat, chemical or pressure sensitive adhesive where the application of light, heat or pressure enhances the adhesion. In yet other embodiments, the adhesive material is adhered to the substrate, solid support, gasket, or combination thereof with the aid of plasma activation of the surface, such as air or oxygen.

The fluid inlet and outlet ports are made by the adhesive material extending beyond the edge of the substrate. In certain embodiments, the adhesive material on the outside of the substrate may be capped with a gasket material. The gasket material may be composed of, but not limited to, silicone, thermoplastic elastomer, adhesive tape, rubber or plastic. The fluid inlet and outlet ports leading into or out of the flow cell may consist of through holes present in the gasket material so that these ports are incorporated into the gasket structure. In still other embodiments, the port can be a different material than the gasket layers and is incorporated into the gasket to provide an open port through the layers.

In certain embodiments, the fluid inlet and outlet ports comprise thin film fluid connectors containing microfluidic channels through which reagents can flow. The connector can be directly coupled to a microfluidic flow cell by a fluidic connection with a laminated planar assembly, which itself provides a small volume, easy-to-use fluidic connection between an external macroscale fluidic device and an assembled flow cell, such as Provide pumps, valves and reservoirs.

In certain embodiments, in addition to microfluidic channels, the membrane can house microvalves that facilitate reagent switching, allowing reagent storage wells to be directly incorporated into or coupled to polymer membranes, and flow cells during particular process steps. To provide isolation. The valve can separate the flow cell to provide a leak tight seal, prevent liquid evaporation, or hold the desired pressure or vacuum level. The valve can further facilitate filling of the flow cell and the flow cell can establish a vacuum prior to opening the valve.

In certain embodiments, the thin film connector may be constructed of flexible plastic having a thickness of generally less than 1 mm. Flexible plastics include, but are not limited to, polyimide films such as Kapton® (DuPont), cyclic olefin copolymers (COC), polyethylene terephthalate (PET), polymethylmethacrylate (PMMA), polycarbonate (PC). , Teflon (registered trademark) (DuPont) and other polytetrafluoroethylene (PTFE), polystyrene (PS), polyethylene (PE), polysulfone (polysuflone) (PSU), polyvinyl chloride (PVC), polyether ether ketone (PEEK) ), polypropylene (PP), heat or pressure sensitive adhesives, thermoplastic elastomers or silicone elastomer films. In certain embodiments, the plastic has sufficient chemical resistance to resist degradation by the reagents used.

In certain embodiments, the connector is made up of multiple plastic layers that are laminated together to form the desired microfluidic feature. In certain embodiments, one or more of these layers have preformed slots or cut out or formed grooves that create encapsulated microfluidic channels once the layers are laminated together. These layers may be directly heat bonded together or use adhesive interlayers including, but not limited to, pressure sensitive adhesives, B-stage adhesives, glues, chemical interlayers or combinations thereof. Or they may be combined with each other. The chemical intermediate layer can be, for example, a chemical primer used in the lamina and can be covalently attached to the surface.

FIG. 3 shows an assembly using one such laminate (300). As shown, the channel laminate is formed by various layers of Kapton®, such as top and bottom layers (310), pressure sensitive adhesive (320) and silicone gasket (330). The channels may be present in the preformed layer (340). The fixture (350) can be used to align the individual layers during assembly, allowing reproducibility of alignment and tight tolerances. This results in a fluid channel encapsulated within a polymeric thin film connector (360) that contains fluid ports in fluid communication with the channel.

As shown in FIG. 4, in certain embodiments, the thin film connector may first be bonded to the flow cell gasket material (410) (FIG. 4A) as part of the subassembly (420). The resulting subassembly can then be adhered to the biological sample (430) (FIG. 4B) to form the microfluidic flow cell (440). In certain other embodiments, the membrane connector may be part of the gasket material itself, in which case the gasket extends beyond the flow cell subassembly as a single component construction, or Some or form part of a laminate containing microfluidic channels.

In certain embodiments, the adhesive interlayer may optionally have a microfluidic feature that cuts through the interlayer. In certain embodiments, valves can be formed by a combination of silicone membranes and corresponding microfluidic channels and valve seats.

In certain embodiments, well-developed methods for building thin film electronic devices can be combined with methods for building microfluidic channels to achieve integration of fluidic and electronic devices, heating/cooling, generation of electromagnetic waves/ Enables conversion/sensing, pressure/vacuum generation/conversion/sensing and flow/electricity sensing elements.

In certain embodiments, the thin film fluid connector may be directly bonded to the silicone gasket layer of the consumable microfluidic flow cell. Alternatively, the thin film fluid connector may be bonded to the ends of the flow cell coverslip with a pressure sensitive adhesive, eliminating the need for a flow cell gasket layer.

In certain embodiments, the alignment features of the thin film connector allow for easy registration in the instrument, such as when it was mounted on the microscope. This connector allows for separation between the fluid seal and the flow cell mount, enhancing ease of use.

In certain embodiments, the thin film connector is designed to allow another fluid connection. One port that functions as a single connection may be used, and other designs may allow multiple inlet and outlet ports. In other embodiments, this port interfaces with a flow channel or valve used to direct or manage flow. In yet another embodiment, as shown in FIG. 5, the connector is configured with an integrated reagent well (510) and valve (520) to provide multiple inlets and flow of reagents into a minimally diluted flow cell. To do.

In certain embodiments, the valve may be incorporated directly into the membrane connector or in fluid connection with the connector to control the flow of reagents into and out of the flow cell.

In certain embodiments, electrical traces can be incorporated into polymer thin films and can be sensing and heating elements and electronic elements that are intimately coupled to the fluid channels described above, such as heating/cooling, electromagnetic waves, generation/transformation/sensing. It enables pressure/vacuum generation/conversion sensing and flow/electric sensing elements. Thin film detectors, such as OLEDs, may also be incorporated for light sensing and detection.

In certain embodiments, the membrane may be flexible, allowing the three-dimensional fluid path and providing non-uniform gap filling between connections or locations of connections. This allows for switching or insertion between different devices of different sizes, which is shown in FIG. 6, where the fluid module (eg, flow cell) is bent (A), twisted ( B), or arranged in series and stacked to allow for multiple modules (C) further arranged to be arranged by thin film fluid connections, which include, but are not limited to, 180°. Including the connection of.

As shown in FIGS. 7A and 7B, in certain other embodiments, the gasket layer (130) can function as a valve that blocks the flow of reagents between the port (140) and the assembled flow cell (160). Provides isolation of the contents within. The valve is closed by deforming the gasket so that it contacts the solid support (150) in the channel region (170) between the substrate and port (140) (FIG. 7C). This deformation can be induced by methods that include, but are not limited to, the movement of pushing a solid structure against the gasket and locally applying air pressure to a particular location on the gasket. Other methods of deforming the gasket and sealing the gasket to the solid support may also be used.

FIG. 8A illustrates one embodiment in which a fluid connection is achieved, wherein the fluid connection fitting further comprises a ring or sealing device fitting with height (200). As shown, the microfluidic flow cell is contacted with the elevated sealing surface (190) to form a seal against the gasket layer (FIG. 8B). The ledge (195) on the fixture can limit the gasket compression distance and provides a leveling surface that allows the solid support to be recorded by an external entity such as the microscope objective of the imager.

FIG. 9A is a view of one of the height sealing devices showing the gasket (130) and ledge (195) in greater detail. The gasket (130) is placed against a raised surface.

In certain embodiments, the amount of compression is determined by the thickness of the gasket and the distance between the tall seal and the ledge. For example, if the total thickness of the assembled flow cell was about 525 μm and the distance between the seal and the ledge was about 500 μm, then the compression distance would be designed to be about 25 μm.

In another embodiment, the compression tolerance can be reduced by forming a seal that completely surrounds the gasket area above the channel formed in the adhesive layer (FIG. 9B), which causes the gasket to face the solid support. Bending can be prevented. This is illustrated in Figure 9B, where the flow channels may be formed with the gasket layer (130) as the bottom surface. This allows variable compression due to manufacturing tolerances without adversely affecting the flow resistance of the connector.

In certain embodiments, the gasket is designed to be compressed by at least 5 μm and 30 μm or less. In other embodiments, the compression distance is designed to be at least 5 μm, but the maximum distance can be up to 200 μm if compression does not deform the gasket. As shown in the figure, in order to make a strong seal between the gasket and the fluid connector, it is desirable that the gasket be compressed at a distance greater than the roughness/variation of the gasket surface. In some embodiments, the gasket should not be compressed so strongly that the gasket deforms and seals against the solid support surface (blocking channels extending beyond the substrate). The compression of the gasket should preferably be less than half the height of the channel. Assuming that the gasket surface roughness is <1 μm after molding using a very smooth master mold, thickness/surface variation is the dominant parameter. Variations in gasket thickness may be on the order of ±5-10 μm, and similar tolerances are expected for fluid connector step sizes. In certain embodiments, the target gasket may be defined at 25 μm±12.5 μm.

In certain embodiments, a solid support (150) biological sample may be included. In certain embodiments, the bond strength of the subassembly (100) with the substrate (120) may be sufficient if the assembly device (160) does not need to be clamped to maintain a seal. The required cohesive strength will depend on the pressure drop that occurs under normal flow conditions, the flexibility of the substrate and solid support, the normal operating temperature and the chemicals in contact with the subassembly material and solid support.

The height of the subassembly (100) and associated assembly flow cell (160) can be determined based on the thickness of the sample. If the sample is a tissue section, the sample will have a thickness of between about 1 μm and about 100 μm. In some embodiments, the tissue section may occupy an area of up to 25 mm x 50 mm. This results in a small in-cell volume or subassembly holding volume in the range of 1 μL to 1000 μL, preferably 25 μL to 200 μL, and is determined by the dimensions of the subassembly. The subassemblies can be designed differently depending on the size of the sample to minimize the internal cell volume while still enclosing the sample. In certain embodiments, dimensional tolerances are believed to be associated with compatible automated equipment or reagent volume adjustments. For example, in certain embodiments, the wall width or height dimensional tolerance may be ±10 μm. In other embodiments, the tolerance may be ±6.25 μm, and in yet other embodiments, the tolerance may be ±5 μm. Tolerances can further aid the use of automated equipment.

If the subassembly (100) is mechanically flexible, the subassembly (100) may bend as fluid flows through the assembly flow cell. The cause of mechanical flexibility is due to the adhesive layer, gasket, substrate or combinations thereof. For example, in certain embodiments, the substrate (120) may be a soft flexible polymer film or glass having a sufficient flexural modulus to deform without breaking. In other embodiments, the gasket may be overmolded onto a glass coverslip, which then becomes the substrate layer (120).

Thus, in certain embodiments, the substrate can function as a layer of flexible material. When the flow is induced with positive pressure, the layer of flexible material bends away from the solid support, effectively creating a larger chamber volume in the center of the flow cell. In these cases, the flow resistance is smaller in the center and more flow occurs in the center. When the flow is induced with negative pressure, the flexible material layer bends towards the solid support, effectively creating a smaller chamber volume in the center of the flow cell. This means that the outer edge of the flow cell has a smaller flow resistance and more flow occurs in these areas. Therefore, the flexible material may act as a flow controller. In certain embodiments, the solid support (150) is flexible and can be functional in a similar fashion.

In certain embodiments, this switchable flow resistance can be used to ensure forward and backward flow with continuous positive and negative pressure to ensure uniform fluid delivery throughout the flow cell. The fluid preferentially flows in the central region of the flow cell and then preferentially at the outer edge of the cell. This is particularly useful for molecular pathology applications where tissue staining is performed in a very wide flow cell and uniform staining is heavy.

The switchable flow resistance can also be used to ensure that no bubbles enter the center of the flow cell. For example, when making a fluid connection between a flow cell and a fluid delivery, air may be introduced at the connection interface. After making such a connection, negative pressure flow causes all the air in the system to flow along the outer edge of the flow cell and then out of the cell. If the substrate was not flexible, air would likely enter the center of the flow cell and may be trapped depending on the cell size and flow characteristics. These bubbles block fluid flow and can prevent uniform fluid delivery to the contents within the cell. In addition, the use of mechanically flexible materials regulates changes in flow resistance throughout the flow cell, facilitating more uniform fluid delivery and system startup and prevention of air entrapment in the cell. Flow can be achieved.

In certain embodiments, the flexibility of the substrate and/or gasket that acts as a layer of flexible material or lid further provides a way to provide optimal high magnification imaging within a microfluidic flow cell. Thus, in a preferred embodiment, the substrate is transparent for imaging in the appropriate wavelength range, for example when the substrate comprises a coverslip or coverslip material. Thus, the flow chamber is obtained with a flexible, permeable lid that is pulled down against the sample to simulate a conventional coverslip solid support and allow for imaging of the image without damaging or removing the microfluidic chamber. The quality can be improved.

In certain embodiments, the layer of flexible material is configured to be pushed away from the sample or bend toward the sample when the chamber is pressurized or evacuated. This is readily accomplished by the addition or removal of fluids, gases or liquids from the flow cell chamber, which is typical of microfluidic systems. With an inlet pump and an outlet pump, these pumps can be operated in unison to flow liquid without significantly affecting the curvature of the lid. If bending is desired, the pump can be made to operate at mismatched speeds or in opposite directions to obtain positive or negative curvature. A chamber under negative pressure is used to pull the lid down (or against) the sample, reducing the height of fluid between the sample and the lid. This reduces the depth of fluid imaged by the imaging system and improves image quality. At certain resolutions/contrasts, imaging of microfluidic channels may suffer from light scattering in the fluid and suboptimal optical coupling. Thus, the flexible lid creates a microfluidic chamber across the sample while allowing the fluid to flow normally during imaging to further reduce the total height of the fluid in the flow chamber.

FIG. 10 shows the result of imaging a human tissue section with an objective lens of ×20. Serial images show the progression at various time points from a 50 μm convex overfilled flow cell to a -50 μm concave underfilled flow cell. The measurement is about z-height measured by changing the focus of the microscope. The zero point corresponds to a nearly flat flow cell glass surface. As shown, the image quality changes based on the pressure change in the chamber that affects the height of the liquid between the sample and the flexible lid.

In certain embodiments, the coverslip may be made of glass, such as silicate glass or borosilicate glass, or specialty plastics such as NUNC™ brand Thermanox® coverslips that have precise light transmission. .. Quartz glass coverslips can also be used if UV transmission is required, for example for fluorescence microscopy.

In certain embodiments, the deformation of the flexible lid depends on the volumetric capacity of the flow cell. In certain embodiments, the flexing of the lid may be related to the material used, for example when the material is silicone, the material acts as a flexible permeable bag to provide significant flexibility. .. In a particular embodiment, the deflection of the lid is about ±200 μm. In a preferred embodiment, the deflection is between −50 and +200 μm, most preferably between −50 and +100 μm, which may result in a z-height change of −20 to +50 μm. Therefore, the deformation of the flexible lid minimizes the height of the fluid in the flow chamber and thus minimizes the spherical aberration caused by the fluid, thus providing optimal imaging of the sample (maximum resolution and contrast). enable.

In certain embodiments, a preferred variation is that there is little liquid between the sample and the flexible lid. Thus, in one embodiment, the deflection is in the range of adhesion layer thickness-sample thickness (about 20 um for a 25 um high chamber and 4 um tissue section).

In certain embodiments, the assembled flow cell (160) can be aligned with a plastic cartridge that can contain a fluid that can flow in the chamber or a dry reagent that is rehydrated. In certain molecular pathology applications, such reagent cartridges can be used to house specific panels pre-packed with biomarkers for specific tests. The cartridge can also be designed to add user-customized reagents.

In certain embodiments, the assembled flow cell comprises a subassembly and a solid support, the solid support carrying a biological sample. The sample is completely enclosed by the subassembly bound to the solid support. In certain embodiments, gluing results in a subassembly permanently or semi-permanently bonded to a solid support, whereby removal of the subassembly consumes the flow cell and a new flow cell for further analysis of the sample. Assembly may be required. Although the flow cell does not need to be reusable, the simple device structure allows low cost manufacturing, as it can be a consumable part that remains attached to a single solid support.

In certain embodiments, the flow cell comprises one or more attachment points configured to match attachment points of another part of the device, such as an imager stage, eg, microscope, thermal management system or fluidic device. In certain embodiments, the attachment point is configured to align with the flow cell and the objective lens of the imager.

In certain embodiments, the resulting flow cell encapsulating the biological sample may be exposed to various reagents and imaging steps. In certain embodiments, the flow cell containing the encapsulated sample may be stored as is for subsequent analysis, or for post hoc analysis after the initial steps. In other embodiments, the subassembly may be adhered to a solid support that carries materials other than biological samples. For example, the solid support may include, but is not limited to, chemicals, mechanical structures or combinations thereof. In certain embodiments, the material may be surface-bound chemicals, polymers and mechanical structures such as, but not limited to, microelectromechanical sensors, actuators and flow blocking elements. The resulting microfluidic flow cell can then be used for analysis or functionalization of the contained material.

In certain embodiments, the flow cell includes, but is not limited to, DNA analysis or amplification, RNA analysis or amplification, nucleic acid sequencing, protein analysis, antigen retrieval, hematoxylin and eosin staining (H&E), immunofluorescent staining. It can be used for tissue or cell analysis, including (IF), immunohistochemical staining (IHC), fluorescence in situ hybridization (FISH) or other histological and morphological staining techniques.

In certain embodiments, DNA fluorescence in situ hybridization (FISH) or RNA FISH on tissue sections can be performed in a microfluidic chamber. Thus, in certain embodiments, an adhesive flow cell is applied to a tissue slide to create a microfluidic chamber. FISH is then performed on the tissue by flowing the reagents, optionally sealing the flow cell (to allow evaporation prevention or pressurization) and optionally adjusting the temperature of the flow cell. After this step is complete, the chamber is optionally filled with encapsulant and the tissue can be imaged in the flow cell. The flow cell remains on the tissue slide for subsequent process steps or storage.

The flow cell creates a reliable, airtight chamber in a consistent and safe way to the tissue. This approach also does not require subsequent chamber removal. The flow cell can be used to automate a fluidic device to perform a FISH process, an example of which is shown in the process map of FIG. This step includes, but is not limited to, water rinse (A), pepsin digestion (B), water rinse (C), PBS wash (D), formalin fixation (E), PBS+MgCL2 wash (F), PBS. Wash (G), alcohol dehydration (H), probe incubation in various conditions (I and J), subsequent wash with solution of SSC and NP40 (K), DAPI staining (L), SSC wash (M) and imaging. (N) is included.

Thus, in another embodiment, a general method for analysis of a biological sample bound to a microfluidic flow solid support is: (a) sample and reagents corresponding to techniques involving histology, morphology or molecular analysis. Are contacted by flowing a reagent through a fluid connector, (b) detecting a signal from a biological sample, and (c) analyzing histological, morphological or molecular components of the sample. ,including. In other embodiments, the signal from the sample may be inactivated and this process step repeated.

Experiments were performed with tissue microarrays enclosed in a microfluidic flow cell and matched to a semi-automatic thermofluidic system. The whole DNA FISH process has been successfully performed in a microfluidic flow cell and validated using this system.

Microfluidic flow cells bring automation and repeatability benefits to immunohistochemistry (IHC) processing, and therefore the same benefits apply to DNA FISH. Compared to IHC automation in microfluidic flow cells, DNA FISH automation is more complicated due to heat and the addition of a wide variety of reagents.

The DNA FISH step involved baking slides for 1 hour at 60° C., then cleaning slides and hydrating and antigen-stimulating sample preparation prior to flow cell assembly. The DNA FISH protocol in the flow cell was optimized for the Her2/CEP17 gene marker of the Vysis commercial probe supplied by Abbott Laboratories.

The flow cell protocol included purging and starting steps between each step to minimize reagent dilution and avoid bubble trapping. The purge and start-up steps include suctioning from the inlet of the flow cell until there is no liquid in the tubing line and manually using a CO ₂ air gun to purge air from the flow cell and leaving any liquid residue at the outlet. extrusion, the step of the CO ₂ air gun using manually purge air from the outlet tubing, and, comprising the step of starting the flow cell by the following reagents. Purging with CO ₂ followed by rapid startup prevents entrapment of bubbles due to the higher solubility of CO ₂ bubbles in water compared to bubbles. Automation of CO2 purging would be apparent to one of ordinary skill in the art.

The experimental set up consisted of a syringe pump for forward fluid flow in conjunction with the heating system. Each reagent was allowed to flow continuously according to the protocol at the specified flow rate for the specified time. The flow cell was then removed from the flow configuration (further described below), the reagent syringe was replaced with the next reagent, and system tubing was restarted. The flow cell is emptied by first withdrawing the liquid (applying suction using a syringe), then purging the flow cell with CO ₂ to force the liquid out of the system and removing the atmosphere in the flow cell from CO ₂ (more easily to water). To minimize air bubbles trapped in the flow cell). This approach was not automatic, but was used to minimize reagent dilution because reagent dilution requires a larger reagent volume to completely replace one reagent with another inside the flow cell.

It is noteworthy that bubbling of water under pressure can result in the formation of bubbles in the cell. FISH probes are usually diluted in a special buffer consisting of a mixture of formamide, dextran and citrate buffered saline (SSC). Such a buffer has a higher boiling point than water, which should prevent boiling during DNA FISH incubation at 80°C. The flow cell design prevents evaporation of solution components.

The flow cell used for the demonstration of DNA FISH is shown in FIG. These flow cells incorporated a thin film fluid connector for easy connection to tubing for reagent flow. In this case, the connection to the pump system was made by an opening and closing magnetic connection between the tubing and the thin film connector. The connector has an inlet, a thin microfluidic channel manufactured by Kapton and an adhesive layer, and an outlet coupled to a flow cell. The inlet has a thin silicone layer that acts as a gasket. Magnets (about 2,000-2,700 Gauss) are placed on each side of the connector to attract each other to clamp the connector tightly and form a tight seal with the help of the gasket layer.

The magnetic auxiliary fluid connection consists of a snug fit small inside diameter tubing and an inner bond of an annular magnet. These connectors are placed against the inlet side of the thin film connector, with smaller cylindrical magnets placed on the opposite side of the thin film connector, as shown in FIG. 12A. These magnets attract and bond the tubing to the thin film connector, creating a tight seal between the two. The magnet is also used to seal the chamber, as shown in Figure 12B. In this case, a small solid cylindrical magnet with similar magnetic strength is used to create a tight seal for the inlet and outlet and prevent any fluid leakage. This approach was used for DNA FISH.

The image shown in FIG. 13 demonstrates the success of the DNA FISH experiment in a microfluidic flow cell, starting with digestion and ending with application of encapsulant followed by imaging.

As shown in FIG. 13, the images include DAPI, CERP17 and Her2 staining. The nuclei appeared to be properly digested, the FISH probe hybridized correctly, and fluorescent signals were present on both CEP17 and Her2 channels.

While this invention has been described in terms of a preferred embodiment, it is recognized that equivalents, alternatives and modifications, in addition to those expressly stated, are within the scope of the appended claims. ..

100 Subassembly Device, Subassembly 110 Adhesive Layer 120 Substrate Layer, Substrate 130 Gasket, Gasket Layer 140 Fluid Inlet/Outlet Port 150 Solid Support 160 Microfluidic Flow Cell, Assembly Flow Cell, Assembly Device 170 Channel Region 190 Overhanging Sealing Surface 195 Ledge 200 Sealing Device Fixture 300 Laminate, Laminate Layer 310 Top and Bottom Layer 320 Pressure Sensitive Adhesive 330 Silicone Gasket 340 Preformed Layer 350 Assembly Fixture 360 Polymeric Thin Film Connector 410 Flow Cell Gasket Material 420 Subassembly 430 Solid Support 440 Microfluidic Flow cell 510 Reagent well 520 Valve

Claims (16)

A microfluidic flow cell, the flow cell comprising:
a) a microfluidic subassembly, the subassembly comprising:
A laminated planar assembly comprising an adhesive layer, a transparent substrate layer, and a gasket layer,
The substrate layer is connected to the gasket layer on one side and the adhesive layer on the other side, the adhesive layer and the gasket layer extending beyond the range of the substrate layer, the gasket layer and the adhesive A laminated planar assembly, wherein at least one of the layers comprises at least one opening, and a fluid inlet port and an outlet port disposed on the gasket layer and/or the adhesive layer at a position outside the boundary of the substrate layers, and an inlet port A subassembly comprising one pump connected to the pump and one pump connected to the outlet port;
b) a solid support adhered to the microfluidic subassembly,
Equipped with
The substrate layer bends away from the solid support by applying a positive pressure through the addition or removal of fluid from the chamber by the pump, and the solid support by applying a negative pressure. bend facing, and that from the center point try Deflection in the range of ± 200 [mu] m, which is configured to adjust the quality of the captured image of the sample being supported on the solid support, and the substrate layer and the solid support A flow cell that forms a flexible, transparent lid of the chamber between. The flow cell of claim 1, wherein the transparent substrate layer comprises glass, plastic or a combination thereof. The flow cell of claim 1, wherein the flexible transparent lid is configured to flex in the range of -50 to +100 μm. The flow cell of claim 1, wherein the flexible transparent lid is configured to flex in the range of -20 to +50 μm. The flow cell of claim 1, wherein the internal dimension has a volumetric volume in the range of 1 μL to 1000 μL. The flow cell according to claim 5, wherein the volume capacity is in the range of 25 μL to 200 μL. The flow cell of claim 1, wherein the fluid port is a thin film fluid connector that includes one or more microfluidic channels in fluid connection with the laminated planar assembly and is located outside the boundaries of the substrate layers. The flow cell of claim 7, wherein the thin film fluid connector is connected to the gasket layer. 9. The flow cell of claim 8, wherein the fluid port further comprises a valve that regulates fluid flow and pressure in the microfluidic flow cell and is connectable to a fluid delivery system. 9. The flow cell of claim 8, further comprising one or more attachment points configured to match the attachment points of the imaging device. The flow cell of claim 10, wherein the one or more attachment points are configured to align the flow cell with an objective lens of an imager. A method for adjusting the quality of a captured image of a sample carried on a solid support of a flow cell according to claim 11.
A method comprising aligning a flow cell with an objective lens of an imaging device, and adjusting the amount of liquid in the flow cell to deflect the flexible lid within a range of ±200 μm from its center point. 13. The method of claim 12, wherein the flexible transparent lid is configured to flex in the range -50 to +100 [mu]m. 14. The method of claim 13, wherein the flexible transparent lid is configured to flex in the range of -20 to +50 [mu]m. 13. The method of claim 12, wherein the sample is a biological sample. 13. The method of claim 12, further comprising capturing an image of the sample using an image capture device.